Antibodies that bind receptor protein designated 2F1

ABSTRACT

2F1 polypeptides are provided, along with DNA sequences, expression vectors and transformed host cells useful in producing the polypeptides. Soluble 2F1 polypeptides find use in inhibiting prostaglandin synthesis and treating inflammation.

BACKGROUND OF THE INVENTION

[0001] The type I interleukin-1 receptor (IL-1RI) mediates thebiological effects of interleukin-1, a pro-inflammatory cytokine (Simset al., Science 241:585-589, 1988; Curtis et al., Proc. Natl. Acad. Sci.USA 86:3045-3049, 1989). A second interleukin-1 receptor (designatedtype II IL-1R or IL-1RII) binds IL-1, but does not appear to mediatesignal transduction (McMahan et al., EMBO J. 10:2821, 1991; Sims et al.,Proc. Natl. Acad. Sci. USA 90:6155-6159, 1993). IL-1RI and IL-1RII eachbind IL-1α and IL-1β.

[0002] IL-1RI and IL-1RII belong to a family of proteins that exhibitsignificant sequence homology. One such protein is IL-1R accessoryprotein (IL-1R AcP), described in Greenfeder et al. (J. Biol. Chem. 270:13757-13765, 1995). This protein, by itself, is not capable of bindingIL-1, but does form a complex with IL-1RI and either IL-1α and IL-1β.When co-expressed with IL-1-RI, recombinant IL-1R AcP increases thebinding affinity of IL-1RI for IL-1β (Greenfeder et al., supra).

[0003] The protein variously known as ST2, ST2L, T1, or Fit-1 also is amember of the IL-1R family, but does not bind IL-1. Cloning of mouse andrat cDNAs encoding membrane-bound and secreted forms of this protein hasbeen reported (Klemenz et al., Proc. Natl. Acad. Sci. USA 86:5708, 1989;Tominaga, FEBS LETTERS 258:301, 1989; Yanagisawa et al., FEBS LETTERS318:83, 1993; Bergers et al., EMBO J. 13:1176, 1994). Human ST2 cDNA andgenomic clones have been isolated as well (Tominaga et al. Biochimica etBiophysica Acta 1171:215, 1992).

[0004] Other proteins exhibiting significant sequence homology withIL-1RI are murine MyD88 (Lord et al., Oncogene 5: 1095-1097, 1990),human rsc786 (Nomura et al., DNA Res. 1:27-35, 1994), and a number ofDrosophila proteins, the best characterized of which is Toll (Hashimotoet al., Cell 52, 269-279, 1988). The tobacco N gene (Whitham et al.,Cell 78:1101-1115, 1994) is among the additional IL-1R family members.

[0005] MyD88, rsc786, Toll, and the tobacco N gene product containdomains exhibiting significant homology to the cytoplasmic domain of theIL-1RI. The IL-1R AcP and ST2 proteins exhibit sequence similarity toIL-1RI in both their extracellular and cytoplasmic portions. The B16Rprotein of vaccinia virus (Goebel et al., Virology 179:247, 1990)appears to be a viral homolog of IL-1RII.

[0006] Identification of additional receptors of this family isdesirable. Such receptor proteins can be studied to determine whether ornot they bind IL-1, and, if so, whether the receptors play a role inmediating signal transduction. The possible existence of additionalaffinity-converting subunits for receptors of this family can beexplored, as well.

SUMMARY OF THE INVENTION

[0007] The present invention provides a novel receptor proteindesignated 2F1. Both soluble and membrane-bound forms of 2F1 aredisclosed herein. The present invention also provides isolated DNAencoding 2F1 proteins, expression vectors comprising the isolated DNA,and a method for producing 2F1 by cultivating host cells transformedwith the expression vectors under conditions appropriate for expressionof the 2F1 protein. Antibodies directed against 2F1 are also disclosed.2F1 finds use in inhibiting prostaglandin synthesis and alleviatinginflammation.

DETAILED DESCRIPTION OF THE INVENTION

[0008] DNA encoding a novel receptor protein designated 2F1 has beenisolated in accordance with the present invention. Expression vectorscomprising the 2F1 DNA are provided, as well as methods for producingrecombinant 2F1 polypeptides by culuring host cells containing theexpression vectors under conditions appropriate for expression of 2F1,then recovering the expressed 2F1 protein. Purified 2F1 protein is alsoencompassed by the present invention, including soluble forms of theprotein comprising the extracellular domain.

[0009] The present invention also provides 2F1 and immunogenic fragmentsthereof that may be employed as immunogens to generate antibodiesspecific thereto. In one embodiment, the antibodies are monoclonalantibodies.

[0010] Human 2F1 clones were isolated as described in example 1. A human2F1 DNA sequence is presented in SEQ ID NO:1, and the amino acidsequence encoded thereby is presented in SEQ ID NO:2. The proteinincludes a signal peptide (amino acids −19 to −1) followed by anextracellular domain (amino acids 1 to 310), a transmembrane region(amino acids 311 to 332), and a cytoplasmic domain (amino acids 333 to522).

[0011] Mouse 2F1 cDNA was isolated by cross-species hybridization, asdescribed in example 2. The DNA and encoded amino acid sequences of thismouse 2F1 DNA are presented in SEQ ID NO:3 and SEQ ID NO:4. The proteinof SEQ ID NO:4 comprises a signal peptide (amino acids −18 to −1), anextracellular domain (amino acids 1 to 307), a transmembrane region(amino acids 308 to 330), and a cytoplasmic domain (amino acids 331 to519). The mouse and human 2F1 amino acid sequences are 65% identical.

[0012] The amino acid sequence of the 2F1 protein indicates that it is amember of the IL-1 receptor family. Of the known IL-1 receptor familymembers, 2F1 has the highest degree of sequence homology with IL-1Raccessory protein (IL-1R AcP), T1/ST2, and type I IL-1 receptor(IL-1RI). The murine 2F1 amino acid sequence of SEQ ID NO:4 is 31%identical to the amino acid sequence of murine IL-1R AcP, 30% identicalto that of the full length murine T1/ST2, and 27% identical to that ofthe murine IL-1RI. The cytoplasmic domains show slightly greatersequence conservation (36%-44%) than do the extracellular portions(20%-27%).

[0013] The binding assay described in example 3 was conducted todetermine whether 2F1 binds IL-1α, IL-1β, or IL-1 receptor antagonist.Although 2F1 is a member of the IL-1 receptor family, it did not bindany of the three proteins tested.

[0014] Human and mouse 2F1 are within the scope of the presentinvention, as are 2F1 proteins derived from other organisms, includingbut not limited to mammalian species such as rat, bovine, porcine, orvarious non-human primates. DNA encoding 2F1 proteins from additionalorganisms can be identified by cross-species hybridization techniques.Messenger RNAs isolated from various cell types can be screened inNorthern blots to determine a suitable source of mRNA for use in cloning2F1 cDNA from other species.

[0015] The term “2F1” as used herein refers to a genus of polypeptidesthat are substantially homologous to a native 2F1 protein (e.g., theprotein of SEQ ID NO:2 or 4), and which exhibit a biological activity ofa native 2F1 protein. 2F1 proteins of the present invention includemembrane-bound proteins (comprising an extracellular domain, atransmembrane region, and a cytoplasmic domain) as well as truncatedproteins that retain a desired property. Such truncated proteinsinclude, for example, soluble 2F1 comprising only the extracellulardomain or a fragment thereof. Also included are variants of native 2F1proteins, wherein the variants retain a desired biological activity of anative 2F1. Such variants are described in more detail below.

[0016] A 2F1 polypeptide, or fragment or variant thereof, can be testedfor biological activity in any suitable assay. When the cytoplasmicdomain is altered (e.g., truncated, or altered by deletion, addition, orsubstitution of amino acid residues), the 2F1 polypeptide can be testedfor biological activity in a signal transduction assay. Such assaysinclude, but are not limited to, those described in examples 5 to 7below. The altered cytoplasmic domain can be fused to the extracellulardomain of an IL-1 receptor, and the resulting chimeric receptor testedfor the ability to respond to IL-1 by NF-κB activation (see theprocedure in example 5), induction of IL-8 promoter function (example6), or stimulation of prostaglandin E₂ synthesis (example 7). 2F1polypeptides that include an extracellular domain (e.g., soluble 2F1, asdescribed below) can be tested for the ability to inhibit prostaglandinE₂ synthesis in vivo in animal studies. The 2F1 is administered in vivo,and prostaglandin E₂ levels in the animals are measured (before andafter administration of 2F1, and compared to control animals) by anysuitable means, e.g., by ELISA.

[0017] One embodiment of the present invention is directed to soluble2F1 polypeptides. Soluble 2F1 polypeptides comprise all or part of theextracellular domain of a native 2F1, but lack the transmembrane regionthat would cause retention of the polypeptide on a cell membrane. Wheninitially synthesized, soluble 2F1 polypeptides advantageously comprisethe native (or a heterologous) signal peptide to promote secretion, butthe signal peptide is cleaved upon secretion of 2F1 from the cell.

[0018] One use of soluble 2F1 polypeptides is in blocking a biologicalactivity of 2F1. Soluble 2F1 may be administered to a mammal to bind anyendogenous 2F1 ligand(s), thereby inhibiting the binding of such ligandsto endogenous receptors comprising 2F1. In one embodiment, a soluble 2F1polypeptide is administered to treat pain or inflammation by inhibitingprostaglandin synthesis, as discussed in more detail below.

[0019] Soluble 2F1 may be identified (and distinguished from itsnon-soluble membrane-bound counterparts) by separating intact cellswhich express the desired protein from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired protein. The presence of 2F1 in the medium indicates thatthe protein was secreted from the cells and thus is a soluble form ofthe desired protein. Soluble 2F1 may be a naturally-occurring form ofthis protein.

[0020] The use of soluble forms of 2F1 is advantageous for certainapplications. Purification of the proteins from recombinant host cellsis facilitated, since the soluble proteins are secreted from the cells.Further, soluble proteins are generally more suitable for intravenousadministration.

[0021] Examples of soluble 2F1 polypeptides include those comprising theentire extracellular domain of a native 2F1 protein. One suchpolypeptide is a soluble human 2F1 comprising amino acids 1 through 310of SEQ ID NO:2. Another is a soluble murine 2F1 comprising amino acids 1through 307 of SEQ ID NO:4. When initially expressed within a host cell,the soluble polypeptide may additionally comprise one of theheterologous signal peptides described below that is functional withinthe host cells employed. Alternatively, the polypeptide may comprise thenative signal peptide, such that the 2F1 comprises amino acids −19through 310 of SEQ ID NO:2 or amino acids −18 through 307 of SEQ IDNO:4. Soluble 2F1 polypeptides include fragments of the extracellulardomain that retain a desired biological activity. DNA sequences encodingsoluble 2F1 polypeptides are encompassed by the present invention.

[0022] 2F1 fragments, including soluble polypeptides, may be prepared byany of a number of conventional techniques. A desired DNA sequence maybe chemically synthesized using known techniques. DNA fragments also maybe produced by restriction endonuclease digestion of a full lengthcloned DNA sequence, and isolated by electrophoresis on agarose gels.Oligonucleotides that reconstruct the 5′ or 3′ end of a DNA fragment toa desired point may be synthesized. The oligonucleotides may contain arestriction endonuclease cleavage site upstream of the desired codingsequence and position an initiation codon (ATG) at the 5′ terminus ofthe coding sequence. Linkers containing restriction endonucleasecleavage site(s) may be employed to insert the desired DNA fragment intoan expression vector. Alternatively, known mutagenesis techniques may beemployed to insert a stop codon at a desired point, e.g., immediatelydownstream of the codon for the last amino acid of the extracellulardomain.

[0023] As a further alternative, the well known polymerase chainreaction (PCR) procedure may be employed to isolate a DNA sequenceencoding a desired protein fragment. Oligonucleotides that define thetermini of the desired fragment are employed as primers in the reaction.PCR procedures are described, for example, in Saiki et al. (Science239:487, 1988) and in Recombinant DNA Methodology, Wu et al. eds.,Academic Press Inc., San Diego, 1989, pp 189-196.

[0024] Regarding the foregoing discussion of signal peptides and thevarious domains of the 2F1 proteins, the skilled artisan will recognizethat the above-described boundaries of such regions of the proteins areapproximate. For example, although computer programs that predict thesite of cleavage of a signal peptide are available, cleavage can occurat sites other than those predicted. Further, it is recognized that aprotein preparation can comprise a mixture of protein molecules havingdifferent N-terminal amino acids, due to cleavage of the signal peptideat more than one site. In addition, the exact boundaries of atransmembrane region may differ from that predicted by a computerprogram. Such forms of 2F1 that retain a desired biological activity areincluded among the 2F1 polypeptides of the present invention.

[0025] The present invention provides purified 2F1 polypeptides, bothrecombinant and non-recombinant. Variants and derivatives of native 2F1proteins that retain a desired biological activity are also within thescope of the present invention. 2F1 variants may be obtained bymutations of nucleotide sequences coding for native 2F1 polypeptides. A2F1 variant, as referred to herein, is a polypeptide substantiallyhomologous to a native 2F1, but which has an amino acid sequencedifferent from that of a native 2F1 because of one or more deletions,insertions or substitutions.

[0026] The variant amino acid sequence preferably is at least 80%identical to a native 2F1 amino acid sequence, most preferably at least90% identical. The percent identity may be determined, for example, bycomparing sequence information using the GAP computer program, version6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

[0027] DNA encoding such variants is provided by the present inventionas well. Such DNA sequences preferably are at least 80% identical to anative 2F1 DNA sequence, most preferably at least 90% identical. Thepercent identity may be determined using known computer programs, suchas the above-described GAP program.

[0028] Alterations of the native amino acid sequence may be accomplishedby any of a number of known techniques. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

[0029] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Methods of making the alterations set forth above aredisclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, Jan. 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462.

[0030] Variants include conservatively substituted sequences, meaningthat one or more amino acid residues of a native 2F1 is replaced by adifferent residue, but that the conservatively substituted 2F1polypeptide retains a desired biological activity of the native protein.Examples of conservative substitutions include substitution of residuesthat do not alter the secondary or tertiary structure of the protein.

[0031] A given amino acid may be replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gin and Asn.Other conservative substitutions, e.g., involving substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown.

[0032] 2F1 proteins also may be modified to create 2F1 derivatives byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives of 2F1s may be prepared by linking the chemicalmoieties to functional groups on 2F1 amino acid side chains, or at theN-terminus or C-terminus of an 2F1 polypeptide or the extracellulardomain thereof. Other derivatives of 2F1 within the scope of thisinvention include covalent or aggregative conjugates of 2F1s with otherproteins or polypeptides, e.g., N-terminal or C-terminal fusionsproduced by recombinant DNA technology. For example, the conjugate maycomprise a heterologous signal or leader polypeptide sequence at theN-terminus of a 2F1 polypeptide. The signal or leader peptideco-translationally or post-translationally directs transfer of theconjugate from its site of synthesis to a site inside or outside of thecell membrane or cell wall.

[0033] 2F1 polypeptide fusions can comprise peptides added to facilitatepurification and identification of 2F1. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(DYKDDDDK) (SEQ ID NO:5), which is highly antigenic and provides anepitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinant protein.Expression systems useful for fusing the Flag® octapeptide to the N- orC-terminus of a given protein are available from Eastman Kodak Co.,Scientific Imaging Systems Division, New Haven, Conn., as are monoclonalantibodies that bind the octapeptide.

[0034] The present invention further includes 2F1 polypeptides with orwithout associated native-pattern glycosylation. 2F1 expressed in yeastor mammalian expression systems may be similar to or significantlydifferent from a native 2F1 polypeptide in molecular weight andglycosylation pattern, depending upon the choice of expression system.Expression of 2F1 polypeptides in bacterial expression systems, such asE. coli, provides non-glycosylated molecules.

[0035] N-glycosylation sites in the 2F1 extracellular domain can bemodified to preclude glycosylation. N-glycosylation sites in eukaryoticpolypeptides are characterized by an amino acid triplet Asn-X-Y, whereinX is any amino acid except Pro and Y is Ser or Thr. The human 2F1protein extracellular domain contains such triplets at amino acids72-74, 83-85, 131-133, 149-151, 178-180, 184-186, 217-219, and 278-280of SEQ ID NO:2. The murine 2F1 protein contains such triplets at aminoacids 32-34, 53-55, 89-91, 93-95, 116-118, 171-173, 176-178, 182-184,215-217, 277-279, and 460-462 of SEQ ID NO:4. Appropriate modificationsto the nucleotide sequence encoding this triplet will result insubstitutions, additions or deletions that prevent attachment ofcarbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid,for example, is sufficient to inactivate an N-glycosylation site. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846.

[0036] Additional variants are those in which cysteine residues that arenot essential for biological activity are deleted or replaced with otheramino acids, preventing formation of incorrect intramolecular disulfidebridges upon renaturation. As with the other IL-1R family members, theextracellular domain of 2F1 contains three immunoglobulin-like (Ig)domains. Based on alignment of the human 2F1 amino acid sequence withthat of other family members, the cysteines predicted to form thetypical intradomain disulfide bonds of the Ig domains are located atpositions 121, 166, 218, and 279 of SEQ ID NO:2. The first (mostN-terminal) Ig domain includes the first (residue 22) but lacks thesecond cysteine of the pair conserved in other proteins of this family.The first Ig domain of 2F1 thus is predicted to lack the intradomaindisulfide bond that is typical of Ig domains. Like all IL-1R-likeproteins except T1/ST2, mouse and human 2F1 also have a cysteine residuejust a few residues C-terminal to the point of signal peptide cleavage(the cysteine at position 2 of of SEQ ID NO:2 and at position 4 of ofSEQ ID NO:4). 2F1 fragments and variants preferably contain theseconserved cysteines.

[0037] Other variants are prepared by modification of adjacent dibasicamino acid residues to enhance expression in yeast systems in which KEX2protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Human 2F1 contains such KEX2 protease processing sites atamino acids 94-95, 296-297, 345-346, 418-419, and 448-449 of SEQ IDNO:2. Murine 2F1 contains KEX2 protease processing sites at amino acids87-88, 96-97, 231-232, 244-245, 295-296, 339-340, 416-417, 432-433, and446-447 of SEQ ID NO:4. Lys-Lys pairings are considerably lesssusceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg toLys-Lys represents a conservative and preferred approach to inactivatingKEX2 sites.

[0038] Naturally occurring 2F1 variants are also encompassed by thepresent invention. Examples of such variants are proteins that resultfrom alternative mRNA splicing events or from proteolytic cleavage ofthe 2F1 protein, wherein a desired biological activity is retained.Alternative splicing of mRNA may yield a truncated but biologicallyactive 2F1 protein, such as a naturally occurring, soluble form of theprotein, for example. Variations attributable to post-translationalprocessing or proteolysis include, for example, differences in the N- orC-termini upon expression in different types of host cells, due toproteolytic removal of one or more terminal amino acids from the 2F1protein (generally from 1-5 terminal amino acids).

[0039] 2F1 proteins in which differences from the amino acid sequence ofSEQ ID NO:2 are attributable to genetic polymorphism (allelic variationamong individuals producing the protein) are also among the naturallyoccurring variants contemplated herein. The human 2F1 sequence presentedin SEQ ID NO:1 is derived from three cDNA clones from a peripheral bloodlymphocyte library and four PCR clones from the epidermal carcinoma lineKB (see example 1). The codon for alanine 298 is polymorphic, beingpresent in the PBL clones and two of the KB clones, and absent from theother two KB clones. It is also absent from the two mouse clones thatwere derived from an EL4 T cell library. The present invention thusprovides human 2F1 proteins either containing or lacking an alanineresidue at position 298.

[0040] The present invention provides isolated DNA sequences encodingthe novel 2F1 polypeptides disclosed herein. 2F1-encoding DNAencompassed by the present invention includes, for example, cDNA,chemically synthesized DNA, DNA isolated by PCR, genomic DNA, andcombinations thereof. Genomic 2F1 DNA may be isolated by conventionaltechniques, e.g., by using the DNA of SEQ ID NOS:1 or 3, or a fragmentthereof, as a probe in a hybridization procedure.

[0041] Particular embodiments of the present invention are directed toan isolated DNA comprising nucleotides 1 to 1626 of SEQ ID NO:1 (theentire coding region), nucleotides 58 to 1626 of SEQ ID NO:1 (encodingmature human 2F1), nucleotides 381 to 1994 of SEQ ID NO:3 (the entirecoding region), or nucleotides 435 to 1994 of SEQ ID NO:3 (encodingmature murine 2F1). In other embodiments, isolated DNA sequences encodea 2F1 fragment, such as one of the above-described soluble polypeptides.Such DNAs include a DNA comprising nucleotides 1 to 985 of SEQ ID NO:1(which encode amino acids −19 to 310 of SEQ ID NO:2), nucleotides 58 to985 of SEQ ID NO:1 (which encode amino acids 1 to 310 of SEQ ID NO:2),nucleotides 381 to 1355 of SEQ ID NO:3 (which encode amino acids −18 to307 of SEQ ID NO:4), and nucleotides 435 to 1355 of SEQ ID NO:3 (whichencode amino acids 1 to 307 of SEQ ID NO:4). DNAs encoding the variousforms of 2F1 disclosed herein, e.g., 2F1 variants and fusion proteins,are encompassed by the present invention.

[0042] Nucleic acid sequences within the scope of the present inventioninclude isolated DNA and RNA sequences that hybridize to the native 2F1nucleotide sequences disclosed herein under moderately or highlystringent conditions, and which encode biologically active 2F1. Moderatestringency hybridization conditions refer to conditions described in,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989).Conditions of moderate stringency, as defined by Sambrook et al.,include use of a prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0), hybridization at about 55° C. in 5×SSC overnight, followed bywashing at 50-55° C. in 2×SSC, 0.1% SDS. Highly stringent conditionsinclude higher temperatures of hybridization and washing. The skilledartisan will recognize that the temperature and wash solution saltconcentration may be adjusted as necessary according to factors such asthe length of the probe. In one embodiment, highly stringent conditionsinclude hybridization at 68° C. followed by washing in 0.1×SSC/0.1% SDSat 68° C.

[0043] Due to the known degeneracy of the genetic code, wherein morethan one codon can encode the same amino acid, a DNA sequence may varyfrom that presented in SEQ ID NO:1 or 3, and still encode an 2F1 proteinhaving the amino acid sequence of SEQ ID NO:2 or 4, respectively. Suchvariant DNA sequences may result from silent mutations that occur duringPCR amplification, for example. Alternatively, the variant sequence maybe the product of deliberate mutagenesis of a native sequence.

[0044] The present invention thus provides isolated DNA sequencesencoding biologically active 2F1, selected from: (a) DNA derived fromthe coding region of a native mammalian 2F1 gene (e.g., DNA comprisingthe coding region of the nucleotide sequence presented in SEQ ID NO:1 or3); (b) DNA capable of hybridization to a DNA of (a) under moderately orhighly stringent conditions; and (c) DNA which is degenerate as a resultof the genetic code to a DNA defined in (a) or (b). The 2F1 proteinsencoded by such DNA sequences are encompassed by the present invention.

[0045] Examples of 2F1 proteins encoded by DNA that varies from thenative DNA sequence of SEQ ID NO:1 or 3, wherein the variant DNA willhybridize to the native DNA sequence under moderately or highlystringent conditions, include, but are not limited to, 2F1 fragments and2F1 proteins comprising inactivated N-glycosylation site(s) orinactivated KEX2 protease processing site(s). Further examples are 2F1proteins encoded by DNA derived from other mammalian species, whereinthe DNA will hybridize to the human DNA of SEQ ID NO:1 or the mouse DNAof SEQ ID NO:3.

[0046] Purified 2F1 Protein and Uses Thereof

[0047] The present invention provides purified 2F1 polyeptides, whichmay be produced by recombinant expression systems as described below orpurified from naturally occurring cells. Conventional proteinpurification techniques may be employed.

[0048] The desired degree of purity may depend on the intended use ofthe protein. A relatively high degree of purity is desired when theprotein is to be administered in vivo, for example. Advantageously, 2F1polypeptides are purified such that no protein bands corresponding toother (non-2F1) proteins are detected by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to 2F1 protein may bedetected by SDS-PAGE, due to differential glycosylation, variations inpost-translational processing, and the like, as discussed above. Mostpreferably, 2F1 is purified to substantial homogeneity, as indicated bya single protein band upon analysis by SDS-PAGE. The protein band may bevisualized by silver staining, Coomassie blue staining, or (if theprotein is radiolabeled) by autoradiography.

[0049] One process for producing the 2F1 protein comprises culturing ahost cell transformed with an expression vector comprising a DNAsequence that encodes 2F1 under conditions such that 2F1 is expressed.The 2F1 protein is then recovered from culture medium or cell extracts,depending upon the expression system employed. As the skilled artisanwill recognize, procedures for purifying the recombinant 2F1 will varyaccording to such factors as the type of host cells employed and whetheror not the 2F1 is secreted into the culture medium.

[0050] For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify 2F1. Some or all of the foregoingpurification steps, in various combinations, can be employed to providea substantially homogeneous recombinant protein.

[0051] It is also possible to utilize an affinity column comprising anantibody that binds 2F1 to purify 2F1 polypeptides by immunoaffinitychromatography. Example 8 describes a procedure for employing the 2F1protein of the present invention as an immunogen to generate monoclonalantibodies.

[0052] The foregoing chromatography procedures are among those that maybe employed to purify either recombinant or non-recombinant 2F1.Recombinant cell culture enables the production of the protein free ofthose contaminating proteins that may be normally associated with 2F1 asit is found in nature, e.g., on the surface of certain cell types.

[0053] Recombinant protein produced in bacterial culture is usuallyisolated by initial disruption of the host cells, centrifugation,extraction from cell pellets if an insoluble polypeptide, or from thesupernatant fluid if a soluble polypeptide, followed by one or moreconcentration, salting-out, ion exchange, affinity purification or sizeexclusion chromatography steps. Finally, RP-HPLC can be employed forfinal purification steps. Microbial cells can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0054] 2F1 preferably is expressed as a secreted polypeptide to simplifypurification. Secreted recombinant polypeptides from a yeast host cellfermentation can be purified by methods analogous to that disclosed byUrdal et al. (J. Chromatog. 296:171, 1984), which includes twosequential, reversed-phase HPLC steps.

[0055] Conjugates comprising a 2F1 polypeptide and a detectable agentare provided herein. The agent preferably is covalently bound to the 2F1polypeptide. Such conjugates find use in in vitro assays, for example.

[0056] Suitable agents include, but are not limited to, radionuclides,chromophores, fluorescent compounds, enzymes that catalyze acolorimetric or fluorometric reaction, and the like, with the particularagent being chosen according to the intended application. Suitableradionuclides include, but are not limited to, ¹²³I, ¹³¹I, ^(99m)Tc,¹¹¹In, and ⁷⁶Br.

[0057] The agents may be attached to the 2F1 using any of theconventional methods by which such compounds are attached topolypeptides in general. Functional groups on amino acid side chains ofan 2F1 may be reacted with functional groups on a desired agent to formcovalent bonds, for example. The agent may be covalently linked to 2F1via an amide bond, hindered disulfide bond, acid-cleavable linkage, andthe like, which are among the linkages that may be chosen according tosuch factors as the structure of the desired agent. Alternatively, the2F1 or the agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for linking variousmolecules to proteins (Pierce Chemical Company, Rockford Ill.). A numberof techniques for radiolabeling proteins are known. Radionuclide metalsmay be attached to 2F1 using a suitable bifunctional chelating agent,examples of which are described in U.S. Pat. Nos. 4,897,255 and4,965,392.

[0058] As described in example 7, the signaling (cytoplasmic) domain of2F1 transduces a biological signal that stimulates prostaglandin E₂synthesis. Prostaglandins are naturally occurring long-chain hydroxyfatty acids exhibiting biological effects that include, inter alia,mediating pain and inflammation.

[0059] One embodiment of the present invention is directed to the use ofsoluble 2F1 polypeptides to inhibit prostaglandin synthesis. In vivo,signal transduction may be initiated by the the binding of unidentifiedligand(s) to receptors comprising 2F1. Soluble 2F1 is administered to amammal to bind such ligands, thereby inhibiting ligand binding toendogenous cell surface 2F1.

[0060] Soluble 2F1 polypeptides may be administered to a mammal to treatconditions that are mediated by a prostaglandin. A condition is saidherein to be mediated by a prostaglandin when the condition is, at leastin part, caused or exacerbated (directly or indirectly) by aprostaglandin.

[0061] Such conditions include, but are not limited to, inflammationassociated with arthritis (especially rheumatoid arthritis andosteoarthritis), inflammation of the lungs associated with allergy orasthma, adult respiratory distress syndrome, inflammatory bowel disease,and inflammation resulting from injury (especially injury of a joint).The desirability of inhibiting prostaglandins to treat fever, Bartter'ssyndrome, diabetes mellitus, patent ductus arteriosus, and dysmenorrheais discussed in Harrisons's Principles of Internal Medicine, 13thEdition, Vol. 1, Isselbacher et al., Eds., McGraw-Hill, Inc. New York,1994, pp 433-435.

[0062] Prostaglandin E₂ (PGE₂) also has been implicated in boneresorption, including the bone resorption associated with rheumatoidarthritis and periodontal disease (Isselbacher et al., Eds., supra, atpage 434). PGE₂ has been reported to cause increased vascularpermeability, which is an aspect of the inflammatory response that canlead to local edema (Isselbacher et al., Eds., supra, at page 435).Prostaglandins and their role in inflammation are discussed further inPathophysiology: Clinical Concepts of Disease Processes, 3rd Edition,Price and Wilson, Eds., McGraw-Hill Book Company, New York, 1986, pp36-38; and Inflammation: Basic Principles and Clinical Correlates,Second Edition, Galin et al., Eds., Raven Press, New York, 1992.

[0063] Prostaglandins have been suggested to play roles in modulatingthe immune response. PGE₂ can suppress mitogen-induced stimulation ofhuman lymphocytes, for example. Inhibition of PGE₂ thus may bebeneficial in patients in which depressed cellular immunity isattributable, at least in part, to the action of prostaglandins. (SeeIsselbacher et al., Eds., supra, at page 435). Roles for PGE₁ and PGE₂in angiogenesis have also been suggested.

[0064] It is notable that the 2F1 signaling domain transduced a signalthat resulted in activation of the transcription factor NF-κB (seeexample 5). The anti-inflammatory effect of certain drugs(glucocorticoids) is believed to be attributable, at least in part, toinhibition of NF-κB activation (Auphan et al., Science 270:286-290,1995; Marx, ^(Science) 270:232-233, 1995). Soluble 2F1 polypeptides thusmay be used to inhibit NF-κB activation signals transduced via 2F1.

[0065] NF-κB activation has been linked to TNF-induced replication ofhuman immunodeficiency virus (HIV) in infected cells, including T cells(Howard et al., Proc. Natl. Acad. Sci. USA 90:2335-2339, 1993). 2F1 isexpressed on T-cells, and an NF-κB activation signal is transduced bythe 2F1 signaling domain. Thus, soluble 2F1 may be employed to reduceHIV expression in HIV-infected cells. An effective amount of soluble 2F1is administered in vivo to inhibit NF-κB activation that results fromsignaling through 2F1. Any HIV replication that would have resulted fromsuch NF-κB activation is thus diminished.

[0066] Oligomeric Forms of 2F1

[0067] Encompassed by the present invention are oligomers, such asdimers, trimers, or higher oligomers, that contain 2F1. Such oligomersmay be naturally occuring or produced by means such as recombinant DNAtechnology.

[0068] The 2F1 moieties of the oligomer may be soluble 2F1 polypeptides.In certain embodiments, the oligomers comprise from two to four 2F1polypeptides.

[0069] Oligomers may be formed by disulfide bonds between cysteineresidues on different 2F1 polypeptides, or by non-covalent interactionsbetween 2F1 polypeptide chains, for example. In other embodiments,oligomers comprise multiple 2F1 polypeptides joined via covalent ornon-covalent interactions between peptide moieties fused to the 2F1polypeptides. Such peptides may be peptide linkers (spacers), orpeptides that have the property of promoting oligomerization. Leucinezippers and certain polypeptides derived from antibodies are among thepeptides that can promote oligomerization of 2F1 polypeptides attachedthereto, as described in more detail below.

[0070] Preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.,(PNAS USA 88:10535, 1991); Byrn et al., (Nature 344:677, 1990); andHollenbaugh and Aruffo (“Construction of Immunoglobulin FusionProteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11, 1992). In one embodiment of the invention, a 2F1 dimeris created by fusing a 2F1 to the Fc region of an antibody (IgG1). TheFc polypeptide preferably is fused to the C-terminus of a soluble 2F1. Agene fusion encoding the 2F1/Fc fusion protein is inserted into anappropriate expression vector. The 2F1/Fc fusion proteins are expressedin host cells transformed with the recombinant expression vector andallowed to assemble much like antibody molecules, whereupon interchaindisulfide bonds form between Fc polypeptides to yield divalent 2F1. Thedesired dimer may be recovered by conventional procedures, e.g., byaffinity chromatography employing a protein A or protein G column thatwill bind the Fc moieties.

[0071] The term “Fc polypeptide” as used herein includes native andmutein forms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization are also included. One suitable Fc polypeptide isdescribed in PCT application WO 93/10151, hereby incorporated byreference. This Fc polypeptide is a single chain polypeptide extendingfrom the N-terminal hinge region to the native C-terminus (i.e., is anessentially full-length antibody Fc region). Another useful Fcpolypeptide is described in U.S. Pat. No. 5,457,035. The amino acidsequence of the mutein is identical to that of the native Fc sequencepresented in WO 93/10151, except that amino acid 19 has been changedfrom Leu to Ala, amino acid 20 has been changed from Leu to Glu, andamino acid 22 has been changed from Gly to Ala. This mutein Fc exhibitsreduced affinity for Fc receptors. Procedures for preparing a fusionprotein containing an Fc or Fc mutein polypeptide are described furtherin Baum et al. (EMBO J. 13:3992-4001, 1994).

[0072] In other embodiments, 2F1 may be substituted for the variableportion of an antibody heavy or light chain. If fusion proteins are madewith both heavy and light chains of an antibody, it is possible to forman 2F1 oligomer with as many as four 2F1 extracellular regions.

[0073] Another method for preparing oligomeric 2F1 involves use of aleucine zipper. Leucine zipper domains are peptides that promoteoligomerization of the proteins in which they are found. Originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, 1988), leucine zippers have since been found in a variety ofdifferent proteins. Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.Examples of leucine zipper domains suitable for producing solubleoligomeric proteins are those described in PCT application WO 94/10308.Peptides that preferentially form trimers include, for example, theleucine zipper derived from lung surfactant protein D (SPD) described inHoppe et al. (FEBS Letters 344:191, 1994), and U.S. patent applicationSer. No. 08/446,922, hereby incorporated by reference. Recombinantfusion proteins comprising a soluble 2F1 polypeptide fused to a leucinezipper peptide are expressed in suitable host cells, and the resultingsoluble oligomeric 2F1 that forms is recovered from the culturesupernatant.

[0074] As a further alternative, oligomeric 2F1 may be expressed as arecombinant fusion protein, with or without peptide linkers between the2F1 moieties. Suitable peptide linkers are known in the art, and may beemployed according to conventional techniques. Among the suitablepeptide linkers are those described in U.S. Pat. Nos. 4,751,180 and4,935,233, which are hereby incorporated by reference. A DNA sequenceencoding a desired peptide linker may be inserted between, and in thesame reading frame as, the DNA sequences encoding 2F1, using anysuitable conventional technique. In one embodiment, two soluble 2F1polypeptides are joined by a peptide linker.

[0075] The above-described oligomers may be purified by conventionalprotein purification procedures. Immunoaffinity chromatography using anantibody directed against 2F1 may be employed, for example. Oligomerscontaining antibody-derived Fc polypeptides may be purified by affinitychromatography, employing a protein A or protein G column that will bindthe Fc moieties.

[0076] The present invention provides isolated DNA sequences encoding2F1 polypeptides fused to immunoglobin-derived polypeptides. Such DNAsequences may encode a soluble 2F1 fused to an antibody Fc regionpolypeptide, for example. DNA sequences encoding fusion proteinscomprising multiple 2F1 polypeptide moieties are also encompassed by thepresent invention.

[0077] Compositions Comprising 2F1

[0078] The present invention provides compositions (includingpharmaceutical compositions) comprising an effective amount of apurified 2F1 polypeptide and a suitable diluent, excipient, or carrier.2F1 polypeptides administered in vivo preferably are in the form of apharmaceutical composition.

[0079] The compositions of the present invention may contain a 2F1protein in any form described herein, including oligomers, variants,derivatives, and biologically active fragments. In one embodiment of theinvention, the composition comprises a soluble human 2F1 protein.

[0080] 2F1 proteins may be formulated according to known methods thatare used to prepare pharmaceutically useful compositions. Componentsthat are commonly employed in pharmaceutical formulations include thosedescribed in Remington's Pharmaceutical Sciences, 16th ed., 1980, MackPublishing Company.

[0081] 2F1 protein employed in a pharmaceutical composition preferablyis purified such that the 2F1 protein is substantially free of otherproteins of natural or endogenous origin, desirably containing less thanabout 1% by mass of protein contaminants residual of productionprocesses. Such compositions, however, can contain other proteins addedas stabilizers, carriers, excipients or co-therapeutics.

[0082] Components of the compositions will be nontoxic to patients atthe dosages and concentrations employed. Ordinarily, the preparation ofsuch compositions entails combining a mammalian 2F1 polypeptide orderivative thereof with buffers, antioxidants such as ascorbic acid, lowmolecular weight (less than about 10 residues) peptides, proteins, aminoacids, carbohydrates including glucose, sucrose, or dextrans, chelatingagents such as EDTA, glutathione, or other stabilizers and excipients.Neutral buffered saline is one appropriate diluent.

[0083] For therapeutic use, the compositions are administered in amanner and dosage appropriate to the indication and the patient.Administration may be by any suitable route, including but not limitedto continuous infusion, local administration, sustained release fromimplants (gels, membranes, and the like), or intravenous injection.

[0084] Antibodies that Specifically Bind 2F1

[0085] The 2F1 proteins of the present invention, or immunogenicfragments thereof, may be employed in generating antibodies. The presentinvention thus provides antibodies that specifically bind 2F1, i.e., theantibodies bind to 2F1 via the antigen-binding sites of the antibody (asopposed to non-specific binding).

[0086] Polyclonal and monoclonal antibodies may be prepared byconventional techniques. See, for example, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980); and Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988). Production of monoclonal antibodiesthat are immunoreactive with 2F1 is further illustrated in example 8below.

[0087] Antigen-binding fragments of such antibodies, which may beproduced using conventional techniques, are also encompassed by thepresent invention. Examples of such fragments include, but are notlimited to, Fab, F(ab′), and F(ab′)₂ fragments. Antibody fragments andderivatives produced by genetic engineering techniques are alsoprovided.

[0088] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques, and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993).

[0089] Among the uses of the antibodies is use in assays to detect thepresence of 2F1 polypeptides, either in vitro or in vivo. The antibodiesfind further use in purifying 2F1 by immunoaffinity chromatography.Those antibodies that additionally can block transduction of abiological signal through 2F1 may be used to inhibit a biologicalactivity mediated by such signal transduction. Disorders mediated orexacerbated (directly or indirectly) by signaling through 2F1 are thustreated. A therapeutic method involves in vivo administration of anamount of such an antibody that is effective in inhibiting an undesired2F1-mediated biological activity. Such antibodies may be administered toinhibit prostaglandin synthesis, thereby treating one of theabove-described prostaglandin-mediated disorders, for example.

[0090] Pharmaceutical compositions comprising an antibody that isdirected against 2F1, and a suitable, diluent, excipient, or carrier,are provided herein. Suitable components of such compositions are asdescribed above for compositions containing 2F1 proteins.

[0091] Conjugates comprising a diagnostic (detectable) or therapeuticagent attached to the above-described antibodies are provided herein. Inone embodiment, the agent is a radionuclide or drug. Techniques forattaching such agents to antibodies are well known.

[0092] Expression Systems

[0093] The present invention provides recombinant expression vectors forexpression of 2F1, and host cells transformed with the expressionvectors. Any suitable expression system may be employed. The vectorsinclude an 2F1 DNA sequence operably linked to suitable transcriptionalor translational regulatory nucleotide sequences, such as those derivedfrom a mammalian, microbial, viral, or insect gene. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, an RNA ribosomal binding site, and appropriate sequenceswhich control transcription and translation initiation and termination.Nucleotide sequences are operably linked when the regulatory sequencefunctionally relates to the 2F1 DNA sequence. Thus, a promoter isoperably linked to an 2F1 DNA sequence if the promoter controls thetranscription of the 2F1 DNA sequence. An origin of replication, whichconfers the ability to replicate in the desired host cells, and aselection gene by which transformants are identified, are generallyincorporated into the expression vector.

[0094] In addition, sequences encoding appropriate signal peptides thatare not native to the 2F1 gene can be incorporated into expressionvectors. For example, a DNA sequence for a signal peptide (secretoryleader) may be fused in frame to the 5′ end of an 2F1 sequence. A signalpeptide that is functional in the intended host cells enhancesextracellular secretion of the 2F1 polypeptide. The signal peptide iscleaved from the 2F1 polypeptide upon secretion of 2F1 from the cell.

[0095] Suitable host cells for expression of 2F1 polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-freetranslation systems could also be employed to produce 2F1 polypeptidesusing RNAs derived from DNA constructs disclosed herein.

[0096] Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, an 2F1 polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant 2F1 polypeptide.

[0097] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR329 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and an 2F1DNA sequence are inserted into the pBR322 vector.

[0098] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage λ P_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of theλP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9(ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).

[0099] 2F1 alternatively may be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia or Kluyveromyces, may also be employed.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene.

[0100] Suitable promoter sequences for yeast vectors include, amongothers, promoters for metallothionein, 3-phosphoglycerate kinase(Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolyticenzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland etal., Biochem. 17:4900, 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657. Another alternative is the glucose-repressible ADH2 promoterdescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast andE. coli may be constructed by inserting DNA sequences from pBR322 forselection and replication in E. coli (Amp^(r) gene and origin ofreplication) into the above-described yeast vectors.

[0101] The yeast α-factor leader sequence may be employed to directsecretion of the 2F1 polypeptide. The α-factor leader sequence isinserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274.Other leader sequences suitable for facilitating secretion ofrecombinant polypeptides from yeast hosts are known to those of skill inthe art. A leader sequence may be modified near its 3′ end to containone or more restriction sites. This will facilitate fusion of the leadersequence to the structural gene.

[0102] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

[0103] Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

[0104] Mammalian or insect host cell culture systems could also beemployed to express recombinant 2F1 polypeptides. Baculovirus systemsfor production of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include COS cells derived from monkey kidney cells(e.g., the COS-1 cell line ATCC CRL 1650, or the COS-7 line ATCC CRL1651, Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK(ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived from theAfrican green monkey kidney cell line CVI (ATCC CCL 70) as described byMcMahan et al. (EMBO J. 10: 2821, 1991).

[0105] Transcriptional and translational control sequences for mammalianhost cell expression vectors may be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

[0106] Expression vectors for use in mammalian host cells can beconstructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983). A useful system for stable high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A useful high expression vector, PMLSV N1/N4, described by Cosmanet al. (Nature 312:768, 1984) has been deposited as ATCC 39890.Additional mammalian expression vectors are pDC406 (McMahan et al., EMBOJ. 10:2821, 1991); HAV-EO (Dower et al., J. Immunol. 142:4314, 1989);pDC201 (Sims et al., Science 241:585, 1988); pDC302 (Mosley et al.,Cell, 59:335, 1989); and those described in U.S. Pat. No. 5,350,683.Other suitable vectors may be derived from retroviruses.

[0107] In place of the native signal sequence, a heterologous signalsequence may be added, such as the signal sequence for interleukin-7(IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence forinterleukin-2 receptor described in Cosman et al., Nature 312:768(1984); the interleukin-4 receptor signal peptide described in EP367,566; the type I interleukin-1 receptor signal peptide described inU.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signalpeptide described in EP 460,846.

[0108] Nucleic Acids and Uses Thereof

[0109] The 2F1-encoding DNAs disclosed herein find use in the productionof 2F1 polypeptides, as discussed above. DNA and RNA complements of theDNA presented in SEQ ID NOS:1 and 3 are provided herein, along with bothsingle-stranded and double-stranded forms thereof. Fragments of the 2F1nucleotide sequences presented herein are also useful. Such fragmentsdesirably comprise at least about 17 contigous nucleotides of thesequence presented in SEQ ID NO:1 or SEQ ID NO:3, or the complementthereof.

[0110] Among the uses of such 2F1 nucleic acids (including fragments) isuse as a probe. Such probes may be employed in cross-specieshybridization procedures to isolate 2F1 DNA from additional mammalianspecies. As one example, a probe corresponding to the extracellulardomain of 2F1 may be employed. The probes also find use in detecting thepresence of 2F1 nucleic acids in in vitro assays and in such proceduresas Northern and Southern blots. Cell types expressing 2F1 can beidentified. Such procedures are well known, and the skilled artisan canchoose a probe of suitable length, depending on the particular intendedapplication. The probes may be labeled (e.g., with ³²P) by conventionaltechniques.

[0111] 2F1 nucleic acid fragments also find use as primers in polymerasechain reactions (PCR). 5′ and 3′ primers corresponding to the termini ofa desired 2F1 DNA may be employed in isolating and amplifying the DNA,using conventional PCR techniques.

[0112] Other useful fragments of the 2F1 nucleic acids are antisense orsense oligonucleotides comprising a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target 2F1 mRNA(sense) or 2F1 DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of 2F1 cDNA. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 toabout 30 nucleotides. The ability to create an antisense or a senseoligonucleotide based upon a cDNA sequence for a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988and van der Krol et al., BioTechniques 6:958, 1988.

[0113] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that blocktranslation (RNA) or transcription (DNA) by one of several means,including enhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of 2F1 proteins.

[0114] Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences. Otherexamples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10448, and other moieties that increaseaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

[0115] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retroviral vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application WO90/13641).

[0116] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors.

[0117] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0118] The following examples are provided to illustrate particularembodiments, and not to limit the scope of the invention.

EXAMPLE 1 Isolation of cDNA Encoding Human 2F1

[0119] A human 2F1 DNA was isolated by polymerase chain reaction (PCR).The primers employed in the reaction were degenerate oligonucleotidesbased on two regions within the cytoplasmic domain of the type I IL-1R.These two regions are among the motifs that are conserved in the IL-1receptor family.

[0120] Initially, appropriate conditions for PCR amplification with thedegenerate primers were determined by using human and mouse type I IL-1receptors and mouse T1/ST2 cDNA clones as template. Using the conditionsthat were determined to yield an amplification product from each ofthese cDNAs, PCR was conducted using a 500 kb human yeast artificialchromosome (YAC) as template. This YAC, designated CO2133, contains DNAfrom the human chromosome 2q12 region and is known to include the type IIL-1 receptor, part of the type II IL-1 receptor, and ST2 (Sims et al.,Cytokine 7:483-490, 1995).

[0121] The polymerase chain reactions (20 μl) employed 0.5 μl of a 16:1mixture of Taq (Perkin-Elmer) and Vent (New England Biolabs) DNApolymerases and contained 200 pmole of each primer, 200 μM dNTPs and5-10 μl of human YAC CO2133 DNA, partially purified by extraction from apulse-field gel. Cycle conditions were: 5 minutes at 94° C., duringwhich time the DNA polymerase mixture was added; 40 cycles of (1 minuteat 94° C., 3 minutes at 35° C., 1 minute at 72° C.); followed by 10minutes at 72° C. The reaction products were separated byelectrophoresis on a low-melting temperature agarose gel. The bandcontaining material between 90 and 150 bp in length was excised, melted,and 5 μl used as template in a second PCR. The second reaction wasperformed similarly to the first, except that only 20 cycles were run.The reaction products were separated by electrophoresis on an agarosegel. The 90-150 bp fraction was eluted, and the DNA was renderedblunt-ended using T4 DNA polymerase, phosphorylated using T4polynucleotide kinase, heated for 10 minutes at 65° C., ethanolprecipitated, and ligated into a vector designated pCRScript (StratageneCloning Systems, La Jolla, Calif.) in the presence of restriction enzymeSrfI.

[0122]E. coli DH10 cells were transformed with the ligation products.White colonies were picked from Xgal plates, their inserts amplified byPCR using vector primers, and a small amount spotted on nylon filters.The filters were subsequently hybridized at 42° C. in aqueous conditionsto a mixture of ³²P-labelled oligonucleotide probes derived from humanand murine type I IL-1R. Filters were washed at 50° C. in 0.3M NaCl. Thehybridization thus was conducted under conditions of relatively lowstringency.

[0123] Only 5 out of 180 inserts hybridized Random DNA sequencing of 9of the non-hybridizing inserts revealed that they were derived fromyeast DNA. One of the five hybridizing inserts gave a stronghybridization signal, and DNA sequencing revealed it to be amplifiedfrom the type I IL-1R gene. Of the four weakly hybridizing inserts,three came from yeast DNA, and one was found to represent a novel gene,which has been designated 2F1.

[0124] The thus-isolated 2F1 DNA fragment was used to probe a cDNAlibrary prepared from human peripheral blood lymphocytes (PBL), in aneffort to isolate a full-length cDNA clone. Hybridizing clones wereidentified, and three 2F1 cDNA clones were isolated from the PBLlibrary. Four additional 2F1 clones were isolated by PCR from the humanepidermal carcinoma line KB (ATCC CCL 17).

[0125] A human 2F1 DNA sequence was elucidated by sequencing theseclones. The nucleotide sequence of the coding region is presented in SEQID NO:1, and the amino acid sequence encoded thereby is presented in SEQID NO:2. The protein of SEQ ID NO:2 is a type I transmembrane protein,with an N-terminal signal peptide (amino acids −19 to −1) followed by anextracellular domain (amino acids 1 to 329), a transmembrane region(amino acids 330 to 351) and a cytoplasmic domain (amino acids 352 to541). The codon for alanine 298 is polymorphic, being present in the PBLclones and two of the KB clones, and absent from the other two KBclones.

EXAMPLE 2 Isolation of Murine 2F1 cDNA

[0126] cDNA encoding murine 2F1 was isolated by cross-specieshybridization, as follows. Human 2F1 cDNA was used as a probe to screena mouse cDNA library derived from the cell line designated EL4 6.1(MacDonald et al., J. Immunol. 135:3944, 1985), which is a subclone ofthe thymoma cell line EL4 (ATCC TIB 39). A hybridizing clone wasisolated. The nucleotide sequence of this mouse 2F1 cDNA and the aminoacid sequence encoded thereby are presented in SEQ ID NO:3 and SEQ IDNO:4.

[0127] The protein of SEQ ID NO:4 comprises a signal peptide (aminoacids −18 to −1), an extracellular domain (amino acids 1 to 307), atransmembrane region (amino acids 308 to 330), and a cytoplasmic domain(amino acids 331 to 519). The mouse 2F1 amino acid sequence of SEQ IDNO:4 is 65% identical to the human 2F1 amino acid sequence presented inSEQ ID NO:2.

EXAMPLE 3 Binding Assay

[0128] 2F1 is a member of the IL-1 receptor family, as discussed above.Since the 2F1 extracellular domain resembles that of the type I and typeII IL-1 receptors, the ability of 2F1 to bind to IL-1 family members wasinvestigated, as follows.

[0129] 2F1 was tested for the ability to bind IL-1α and IL-1β (March etal. Nature (Lond.) 315:641, 1985), as well as IL-1 receptor antagonistprotein (Eisenberg et al. Nature 343:341, 1990; Hannum et al., Nature343:336, 1990; and Carter et al., Nature 344:633, 1990). IL-1 receptorantagonist (IL-1ra) binds to IL-1 receptors, but does not transduce asignal. By competing with IL-1 for binding to endogenous IL-1 receptors,IL-1ra inhibits biological effects mediated by IL-1.

[0130] A soluble fusion protein designated 2F1/Fc, which comprises thehuman 2F1 extracellular domain joined to the Fc region of a human IgG1,was generated by procedures analogous to those described in Baum et al.(EMBO J. 13:3992-4001, 1994). A soluble type I IL-1RI/Fc fusion proteincomprising the extracellular domain of human type I IL-1R fused to theFc region polypeptide was prepared for use as a positive control.

[0131] A BIAcore biosensor (Pharmacia Biosensor AB, Piscataway, N.J.)was used to examine binding of IL-1 ligands to the human 2F1/Fc fusionprotein, using procedures essentially as described in Arend et al. (J.Immunol. 153: 4766-4774, 1994). Briefly, a goat anti-human IgG serumdirected against the Fc region (Jackson Immunoresearch Laboratories,Inc., West Grove, Pa.), covalently coupled to the dextran matrix of ahydrogel chip, was used to capture the human 2F1/Fc protein. The 2F1/Fcfusion protein thus was immobilized on the BIAcore chip. The three IL-1ligands, at several different concentrations, were reacted with thecaptured protein, and the change of mass per unit area over time wasmeasured.

[0132] The biosensor analysis demonstrated easily measurable binding ofhuman IL-1α, IL-1β, and IL-1ra to the human type I IL-1R/Fc fusionprotein (positive control). However, no binding of any of the three IL-1proteins to the 2F1/Fc fusion protein was detected. Thus, despite itssignificant sequence homology, 2F1 is not an IL-1 receptor.

EXAMPLE 4 Northern Blot Analysis

[0133] The presence of 2F1 mRNA in various cell types was investigatedby Northern blot analysis, using standard techniques. Northern blotspurchased from Clonetech, Palo Alto, Calif., which contain 2 mg of humanpolyA⁺ RNA in each lane, were probed overnight with a ³²P-labeledantisense 2F1 riboprobe at 63° C. in 0.75M NaCl/50% formamide, andwashed at 63° C. in 0.3M NaCl. To ascertain evenness of loading as wellas effectiveness of rRNA removal, the filters were subsequently probedfor GAPDH and 28S rRNA.

[0134] A single hybridizing band, migrating with or slightly faster than28S rRNA, was found in spleen, thymus, leukocyte, liver, lung, heart,small and large intestine, prostate and placenta. It is possible, butuncertain, that a weak signal was seen in testis and ovary. Nohybridizing band was detected in brain, skeletal muscle, kidney, andpancreas.

EXAMPLE 5 Signaling Assay

[0135] The signal transduction capability of the 2F1 cytoplasmic domainwas investigated in the following assay. An expression vector encoding achimeric receptor, in which the extracellular and transmembrane portionsof the mouse type I IL-1 receptor (IL-1RI) were fused to the cytoplasmicportion of the human 2F1, was constructed. The vector encoded aminoacids 1 to 362 of the murine IL-1RI fused to amino acids 332 to 522 ofhuman 2F1. In preparing the construct, a BglII site was introduced intothe murine IL-1RI DNA, just 3′ of the transmembrane region. Thisresulted in the valine residue at position 361 of the murine IL-1RIbeing changed to isoleucine, which is the amino acid present in thehuman IL-1RI at that position.

[0136] Use of the chimeric receptor made it possible to assay forresponse to a known ligand (IL-1). When cells expressing IL-1RI arecontacted with IL-1α or IL-1β, a number of responses are induced,including stimulation of nuclear localization of the transcriptionfactor NF-κB (Thanos, D. and T. Maniatis, Cell 80:529-532, 1995).Activated NF-κB complexes translocate to the nucleus and bind thecognate recognition sequence.

[0137] The chimeric receptor was expressed in COS-7 cells (ATCC CRL1651), and the ability of IL-1 to activate the transcription factorNF-κB was examined in an NF-κB gel assay. A sheep anti-human IL-1RIpolyclonal antiserum (designated P3 herein) was used to block theendogenous (cross-reactive) monkey IL-1R, without affecting IL-1 bindingto the transfected murine IL-1RI/2F1 chimera. COS-7 cells transfectedwith an expression vector encoding the extracellular and transmembraneportions of the murine IL-1RI, but no cytoplasmic domain, were employedas a negative control. As a positive control, COS-7 cells weretransfected with an expression vector encoding full length murineIL-1RI.

[0138] The assay procedure was as follows. COS-7 cells were transfectedwith the receptor constructs. Two days post-transfection, cells weretreated with the blocking antibody and stimulated (30 minutes, 1 ng/ml)with human IL-1α. Immediately after the blocking and IL-1 stimulation,nuclear extracts were prepared from cell samples, essentially asdescribed by Ostrowski et al. (J. Biol. Chem. 266:12722-33, 1991). Adouble-stranded synthetic oligonucleotide probe containing the KBenhancer element from the immunoglobulin k light chain was 5′ endlabelled by phosphorylation with [γ-³²P]ATP. The nuclear extracts (10μg) were incubated with the ³²P-labeled probe for 20 minutes at roomtemperature, and protein-DNA complexes then were resolved byelectrophoresis in 0.5×TBE 10% polyacrylamide gels (Novex). NF-κBcomplexed with DNA indicates NF-κB activation.

[0139] The 2F1 cytoplasmic domain was found to induce NF-κB DNA bindingability, in response to IL-1 stimulation of the chimeric receptormolecule. The induction was comparable in magnitude to that mediated viathe murine IL-1RI (positive control).

EXAMPLE 6 Signaling Assay

[0140] The signalling capability of 2F1 was examined further in aninterleukin-8 (IL-S) promoter activation assay. When cells expressingthe type I IL-1R are contacted with IL-1, transcription of the IL-8 geneis stimulated (Mukaida et al., J. Biol. Chem. 265:21128-33, 1990). TheIL-1R/2F1 chimeric receptor described in example 5 was employed in thisassay, so that response to a known ligand (IL-1) could be investigated.

[0141] A reporter plasmid designated pIL8p, carrying a partial humanIL-8 promoter fused to the coding region of the human IL-2 receptoralpha chain, was prepared. COS7 cells (1×10⁵ cells per well in a 12-welltissue culture plate) were co-transfected with 1500 ng of pIL8p and 500ng of an expression vector encoding the IL-1R/2F1 chimeric receptor.Twenty-four hours post-transfection, the culture medium was changed andthe cells were contacted with a blocking antibody, then stimulated with1 ng/ml human IL-1α or left unstimulated. The blocking antibody was a1:100 dilution of the sheep anti-human IL-1RI polyclonal serum P3 (seeexample 5), which at that concentration blocks binding of IL-1 to theendogenous COS7 cell IL-1 receptors, but has no effect on binding ofIL-1 to the mouse IL-1RI portion of the recombinant chimeric receptor.

[0142] Twelve to sixteen hours post-stimulation, cells were washed twicewith binding medium containing 5% (w/v) non-fat dry milk (5% MBM), andblocked with 2 ml 5% MBM at room temperature for 30 minutes. Cell werethen incubated at room temperature for 60-90 minutes with 1.5 mls/wellof 5% MBM containing 1 μg/ml of mouse monoclonal antibody 2A3 directedagainst IL-2Rα (Cosman et al., Nature 312:768-771, 1984), with gentlerocking. Cells were washed with 5% MBM, then incubated for one hour atroom temperature with 1 ml/well of 5% MBM containing a 1:100 dilution of[¹²⁵1]goat anti-mouse IgG (Sigma Chemical Company, St. Louis, Mo.).Wells were washed four times with 5% MBM, twice with PBS, then strippedby adding 1 ml 0.5 M NaOH, and the counts per minute determined.

[0143] The IL-1R/2F1 chimeric receptor was found to respond to IL-1α byinduction of IL-8 promoter function. Transcription of the reporterconstruct was induced by IL-1 stimulation of the IL-1R/2F1 chimera, toabout half the level mediated by the intact mouse type I IL-1R.

EXAMPLE 7 Prostaglandin Synthesis

[0144] In a third assay, the IL-1R/2F1 chimeric receptor was expressedin KB human epidermal carcinoma cells (ATCC CCL 17). In the presence ofpolyclonal antiserum that blocks human type I IL-1 receptors (seeexample 5), IL-1 stimulation of the chimeric receptor resulted in thesynthesis of prostaglandin E₂.

EXAMPLE 8 Monoclonal Antibodies Directed Against 2F1

[0145] This example illustrates the preparation of monoclonal antibodiesthat are immunoreactive with a 2F1 protein. Human 2F1 is expressed inmammalian host cells, such as COS-7 or CV-1/EBNA-1 cells. The expressed2F1 is purified and employed as an immunogen in generating monoclonalantibodies, using conventional techniques such as those described inU.S. Pat. No. 4,411,993. Alternative immunogens include, but are notlimited to, 2F1 fragments (e.g., soluble 2F1 comprising theextracellular domain), a soluble 2F1/Fc fusion protein, or cellsexpressing recombinant 2F1 on the cell surface.

[0146] Briefly, mice are immunized with 2F1 emulsified in completeFreund's adjuvant and injected subcutaneously or intraperitoneally inamounts ranging from 10-100 μg. Ten to twelve days later, the immunizedanimals are boosted with additional 2F1 emulsified in incompleteFreund's adjuvant. Mice are boosted thereafter on a weekly to bi-weeklyimmunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision for testing by dot blotassay or ELISA (Enzyme-Linked Immunosorbent Assay), for 2F1 antibodies.

[0147] Following detection of an appropriate antibody titer, positiveanimals are provided one last intravenous injection of 2F1 in saline.Three to four days later, the animals are sacrificed, and spleen cellsare harvested and fused to a murine myeloma cell line, e.g., NS1 orpreferably P3x63Ag8.653 (ATCC CRL 1580). Fusions generate hybridomacells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

[0148] The hybridoma cells are screened by ELISA for reactivity againstpurified 2F1 by adaptations of the techniques disclosed in Engvall etal. (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004. Apreferred screening technique is the antibody capture techniquedescribed in Beckmann et al. (J. Immunol. 144-4212, 1990). Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-2F1monoclonal antibodies. Alternatively, hybridoma cells can be grown iiivitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to protein A orprotein G can also be used, as can affinity chromatography based uponbinding to 2F1.

1 5 1626 base pairs nucleic acid single linear cDNA NO NO hu2F1 CDS1..1626 mat_peptide 58..1623 sig_peptide 1..57 1 ATG AAT TGT AGA GAA TTACCC TTG ACC CTT TGG GTG CTT ATA TCT GTA 48 Met Asn Cys Arg Glu Leu ProLeu Thr Leu Trp Val Leu Ile Ser Val -19 -15 -10 -5 AGC ACT GCA GAA TCTTGT ACT TCA CGT CCC CAC ATT ACT GTG GTT GAA 96 Ser Thr Ala Glu Ser CysThr Ser Arg Pro His Ile Thr Val Val Glu 1 5 10 GGG GAA CCT TTC TAT CTGAAA CAT TGC TCG TGT TCA CTT GCA CAT GAG 144 Gly Glu Pro Phe Tyr Leu LysHis Cys Ser Cys Ser Leu Ala His Glu 15 20 25 ATT GAA ACA ACC ACC AAA AGCTGG TAC AAA AGC AGT GGA TCA CAG GAA 192 Ile Glu Thr Thr Thr Lys Ser TrpTyr Lys Ser Ser Gly Ser Gln Glu 30 35 40 45 CAT GTG GAG CTG AAC CCA AGGAGT TCC TCG AGA ATT GCT TTG CAT GAT 240 His Val Glu Leu Asn Pro Arg SerSer Ser Arg Ile Ala Leu His Asp 50 55 60 TGT GTT TTG GAG TTT TGG CCA GTTGAG TTG AAT GAC ACA GGA TCT TAC 288 Cys Val Leu Glu Phe Trp Pro Val GluLeu Asn Asp Thr Gly Ser Tyr 65 70 75 TTT TTC CAA ATG AAA AAT TAT ACT CAGAAA TGG AAA TTA AAT GTC ATC 336 Phe Phe Gln Met Lys Asn Tyr Thr Gln LysTrp Lys Leu Asn Val Ile 80 85 90 AGA AGA AAT AAA CAC AGC TGT TTC ACT GAAAGA CAA GTA ACT AGT AAA 384 Arg Arg Asn Lys His Ser Cys Phe Thr Glu ArgGln Val Thr Ser Lys 95 100 105 ATT GTG GAA GTT AAA AAA TTT TTT CAG ATAACC TGT GAA AAC AGT TAC 432 Ile Val Glu Val Lys Lys Phe Phe Gln Ile ThrCys Glu Asn Ser Tyr 110 115 120 125 TAT CAA ACA CTG GTC AAC AGC ACA TCATTG TAT AAG AAC TGT AAA AAG 480 Tyr Gln Thr Leu Val Asn Ser Thr Ser LeuTyr Lys Asn Cys Lys Lys 130 135 140 CTA CTA CTG GAG AAC AAT AAA AAC CCAACG ATA AAG AAG AAC GCC GAG 528 Leu Leu Leu Glu Asn Asn Lys Asn Pro ThrIle Lys Lys Asn Ala Glu 145 150 155 TTT GAA GAT CAG GGG TAT TAC TCC TGCGTG CAT TTC CTT CAT CAT AAT 576 Phe Glu Asp Gln Gly Tyr Tyr Ser Cys ValHis Phe Leu His His Asn 160 165 170 GGA AAA CTA TTT AAT ATC ACC AAA ACCTTC AAT ATA ACA ATA GTG GAA 624 Gly Lys Leu Phe Asn Ile Thr Lys Thr PheAsn Ile Thr Ile Val Glu 175 180 185 GAT CGC AGT AAT ATA GTT CCG GTT CTTCTT GGA CCA AAG CTT AAC CAT 672 Asp Arg Ser Asn Ile Val Pro Val Leu LeuGly Pro Lys Leu Asn His 190 195 200 205 GTT GCA GTG GAA TTA GGA AAA AACGTA AGG CTC AAC TGC TCT GCT TTG 720 Val Ala Val Glu Leu Gly Lys Asn ValArg Leu Asn Cys Ser Ala Leu 210 215 220 CTG AAT GAA GAG GAT GTA ATT TATTGG ATG TTT GGG GAA GAA AAT GGA 768 Leu Asn Glu Glu Asp Val Ile Tyr TrpMet Phe Gly Glu Glu Asn Gly 225 230 235 TCG GAT CCT AAT ATA CAT GAA GAGAAA GAA ATG AGA ATT ATG ACT CCA 816 Ser Asp Pro Asn Ile His Glu Glu LysGlu Met Arg Ile Met Thr Pro 240 245 250 GAA GGC AAA TGG CAT GCT TCA AAAGTA TTG AGA ATT GAA AAT ATT GGT 864 Glu Gly Lys Trp His Ala Ser Lys ValLeu Arg Ile Glu Asn Ile Gly 255 260 265 GAA AGC AAT CTA AAT GTT TTA TATAAT TGC ACT GTG GCC AGC ACG GGA 912 Glu Ser Asn Leu Asn Val Leu Tyr AsnCys Thr Val Ala Ser Thr Gly 270 275 280 285 GGC ACA GAC ACC AAA AGC TTCATC TTG GTG AGA AAA GCA GAC ATG GCT 960 Gly Thr Asp Thr Lys Ser Phe IleLeu Val Arg Lys Ala Asp Met Ala 290 295 300 GAT ATC CCA GGC CAC GTC TTCACA AGA GGA ATG ATC ATA GCT GTT TTG 1008 Asp Ile Pro Gly His Val Phe ThrArg Gly Met Ile Ile Ala Val Leu 305 310 315 ATC TTG GTG GCA GTA GTG TGCCTA GTG ACT GTG TGT GTC ATT TAT AGA 1056 Ile Leu Val Ala Val Val Cys LeuVal Thr Val Cys Val Ile Tyr Arg 320 325 330 GTT GAC TTG GTT CTA TTT TATAGA CAT TTA ACG AGA AGA GAT GAA ACA 1104 Val Asp Leu Val Leu Phe Tyr ArgHis Leu Thr Arg Arg Asp Glu Thr 335 340 345 TTA ACA GAT GGA AAA ACA TATGAT GCT TTT GTG TCT TAC CTA AAA GAA 1152 Leu Thr Asp Gly Lys Thr Tyr AspAla Phe Val Ser Tyr Leu Lys Glu 350 355 360 365 TGC CGA CCT GAA AAT GGAGAG GAG CAC ACC TTT GCT GTG GAG ATT TTG 1200 Cys Arg Pro Glu Asn Gly GluGlu His Thr Phe Ala Val Glu Ile Leu 370 375 380 CCC AGG GTG TTG GAG AAACAT TTT GGG TAT AAG TTA TGC ATA TTT GAA 1248 Pro Arg Val Leu Glu Lys HisPhe Gly Tyr Lys Leu Cys Ile Phe Glu 385 390 395 AGG GAT GTA GTG CCT GGAGGA GCT GTT GTT GAT GAA ATC CAC TCA CTG 1296 Arg Asp Val Val Pro Gly GlyAla Val Val Asp Glu Ile His Ser Leu 400 405 410 ATA GAG AAA AGC CGA AGACTA ATC ATT GTC CTA AGT AAA AGT TAT ATG 1344 Ile Glu Lys Ser Arg Arg LeuIle Ile Val Leu Ser Lys Ser Tyr Met 415 420 425 TCT AAT GAG GTC AGG TATGAA CTT GAA AGT GGA CTC CAT GAA GCA TTG 1392 Ser Asn Glu Val Arg Tyr GluLeu Glu Ser Gly Leu His Glu Ala Leu 430 435 440 445 GTG GAA AGA AAA ATTAAA ATA ATC TTA ATT GAA TTT ACA CCT GTT ACT 1440 Val Glu Arg Lys Ile LysIle Ile Leu Ile Glu Phe Thr Pro Val Thr 450 455 460 GAC TTC ACA TTC TTGCCC CAA TCA CTA AAG CTT TTG AAA TCT CAC AGA 1488 Asp Phe Thr Phe Leu ProGln Ser Leu Lys Leu Leu Lys Ser His Arg 465 470 475 GTT CTG AAG TGG AAGGCC GAT AAA TCT CTT TCT TAT AAC TCA AGG TTC 1536 Val Leu Lys Trp Lys AlaAsp Lys Ser Leu Ser Tyr Asn Ser Arg Phe 480 485 490 TGG AAG AAC CTT CTTTAC TTA ATG CCT GCA AAA ACA GTC AAG CCA GGT 1584 Trp Lys Asn Leu Leu TyrLeu Met Pro Ala Lys Thr Val Lys Pro Gly 495 500 505 AGA GAC GAA CCG GAAGTC TTG CCT GTT CTT TCC GAG TCT TAA 1626 Arg Asp Glu Pro Glu Val Leu ProVal Leu Ser Glu Ser * 510 515 520 541 amino acids amino acid linearprotein 2 Met Asn Cys Arg Glu Leu Pro Leu Thr Leu Trp Val Leu Ile SerVal -19 -15 -10 -5 Ser Thr Ala Glu Ser Cys Thr Ser Arg Pro His Ile ThrVal Val Glu 1 5 10 Gly Glu Pro Phe Tyr Leu Lys His Cys Ser Cys Ser LeuAla His Glu 15 20 25 Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys Ser Ser GlySer Gln Glu 30 35 40 45 His Val Glu Leu Asn Pro Arg Ser Ser Ser Arg IleAla Leu His Asp 50 55 60 Cys Val Leu Glu Phe Trp Pro Val Glu Leu Asn AspThr Gly Ser Tyr 65 70 75 Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp LysLeu Asn Val Ile 80 85 90 Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg GlnVal Thr Ser Lys 95 100 105 Ile Val Glu Val Lys Lys Phe Phe Gln Ile ThrCys Glu Asn Ser Tyr 110 115 120 125 Tyr Gln Thr Leu Val Asn Ser Thr SerLeu Tyr Lys Asn Cys Lys Lys 130 135 140 Leu Leu Leu Glu Asn Asn Lys AsnPro Thr Ile Lys Lys Asn Ala Glu 145 150 155 Phe Glu Asp Gln Gly Tyr TyrSer Cys Val His Phe Leu His His Asn 160 165 170 Gly Lys Leu Phe Asn IleThr Lys Thr Phe Asn Ile Thr Ile Val Glu 175 180 185 Asp Arg Ser Asn IleVal Pro Val Leu Leu Gly Pro Lys Leu Asn His 190 195 200 205 Val Ala ValGlu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser Ala Leu 210 215 220 Leu AsnGlu Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu Asn Gly 225 230 235 SerAsp Pro Asn Ile His Glu Glu Lys Glu Met Arg Ile Met Thr Pro 240 245 250Glu Gly Lys Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn Ile Gly 255 260265 Glu Ser Asn Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser Thr Gly 270275 280 285 Gly Thr Asp Thr Lys Ser Phe Ile Leu Val Arg Lys Ala Asp MetAla 290 295 300 Asp Ile Pro Gly His Val Phe Thr Arg Gly Met Ile Ile AlaVal Leu 305 310 315 Ile Leu Val Ala Val Val Cys Leu Val Thr Val Cys ValIle Tyr Arg 320 325 330 Val Asp Leu Val Leu Phe Tyr Arg His Leu Thr ArgArg Asp Glu Thr 335 340 345 Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe ValSer Tyr Leu Lys Glu 350 355 360 365 Cys Arg Pro Glu Asn Gly Glu Glu HisThr Phe Ala Val Glu Ile Leu 370 375 380 Pro Arg Val Leu Glu Lys His PheGly Tyr Lys Leu Cys Ile Phe Glu 385 390 395 Arg Asp Val Val Pro Gly GlyAla Val Val Asp Glu Ile His Ser Leu 400 405 410 Ile Glu Lys Ser Arg ArgLeu Ile Ile Val Leu Ser Lys Ser Tyr Met 415 420 425 Ser Asn Glu Val ArgTyr Glu Leu Glu Ser Gly Leu His Glu Ala Leu 430 435 440 445 Val Glu ArgLys Ile Lys Ile Ile Leu Ile Glu Phe Thr Pro Val Thr 450 455 460 Asp PheThr Phe Leu Pro Gln Ser Leu Lys Leu Leu Lys Ser His Arg 465 470 475 ValLeu Lys Trp Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser Arg Phe 480 485 490Trp Lys Asn Leu Leu Tyr Leu Met Pro Ala Lys Thr Val Lys Pro Gly 495 500505 Arg Asp Glu Pro Glu Val Leu Pro Val Leu Ser Glu Ser 510 515 520 2830base pairs nucleic acid single linear cDNA NO NO mu2F1 CDS 381..1994mat_peptide 435..1991 sig_peptide 381..434 3 TCCCAGCCCT CCACCTCCCTACCCCCGGTC GTTGGCTTCT TCTTCTTCTT CTTCTTTTTT 60 TTTTTTCCTG CGATAATTCTCTGGTTTGCC AAATCTCTCT AATCAAGCTC CTGGCCTTGC 120 CTCACTGTGC CTTCCCTCCCTGTCTGTTGT CACAGTTGTG GACCAGGAGG TATTTAGTCT 180 CACTTGCTGG GCGAATCCTGCTTCACAGAT GTAAGCGAAG GAGAAGCCAC TGCCCAGGCC 240 TGTGTGTGGG CCACCTCTCTGAAGGTAAGG GCAGACTCTG ATGTCCAGTC CTCACTGTCT 300 TCTGCTGTCT GGAGCAAGGAGAGGAACCAC CCACAACGAT CCTGAAAACA AGAGATACCA 360 TTCAAAGTGG AAGCCTAAACATG CAT CAT GAA GAA TTA ATC TTG ACA CTC 410 Met His His Glu Glu Leu IleLeu Thr Leu -18 -15 -10 TGC ATT CTC ATT GTT AAA AGT GCC TCA AAA AGT TGTATT CAC CGA TCA 458 Cys Ile Leu Ile Val Lys Ser Ala Ser Lys Ser Cys IleHis Arg Ser -5 1 5 CAA ATT CAT GTG GTA GAG GGA GAA CCT TTT TAT CTG AAGCCA TGT GGC 506 Gln Ile His Val Val Glu Gly Glu Pro Phe Tyr Leu Lys ProCys Gly 10 15 20 ATA TCT GCA CCA GTG CAC AGG AAT GAA ACA GCC ACC ATG AGATGG TTC 554 Ile Ser Ala Pro Val His Arg Asn Glu Thr Ala Thr Met Arg TrpPhe 25 30 35 40 AAA GGC AGT GCT TCA CAT GAG TAT AGA GAG CTG AAC AAC AGAAGC TCG 602 Lys Gly Ser Ala Ser His Glu Tyr Arg Glu Leu Asn Asn Arg SerSer 45 50 55 CCC AGA GTC ACT TTT CAT GAT CAC ACC TTG GAA TTC TGG CCA GTTGAG 650 Pro Arg Val Thr Phe His Asp His Thr Leu Glu Phe Trp Pro Val Glu60 65 70 ATG GAG GAT GAG GGA ACG TAC ATT TCT CAA GTC GGA AAT GAT CGT CGC698 Met Glu Asp Glu Gly Thr Tyr Ile Ser Gln Val Gly Asn Asp Arg Arg 7580 85 AAT TGG ACC TTA AAT GTC ACC AAA AGA AAC AAA CAC AGC TGT TTC TCT746 Asn Trp Thr Leu Asn Val Thr Lys Arg Asn Lys His Ser Cys Phe Ser 9095 100 GAC AAG CTC GTG ACA AGC AGA GAT GTT GAA GTT AAC AAA TCT CTG CAT794 Asp Lys Leu Val Thr Ser Arg Asp Val Glu Val Asn Lys Ser Leu His 105110 115 120 ATC ACT TGT AAG AAT CCT AAC TAT GAA GAG CTG ATC CAG GAC ACATGG 842 Ile Thr Cys Lys Asn Pro Asn Tyr Glu Glu Leu Ile Gln Asp Thr Trp125 130 135 CTG TAT AAG AAC TGT AAG GAA ATA TCC AAA ACC CCA AGG ATC CTGAAG 890 Leu Tyr Lys Asn Cys Lys Glu Ile Ser Lys Thr Pro Arg Ile Leu Lys140 145 150 GAT GCC GAG TTT GGA GAT GAG GGC TAC TAC TCC TGC GTG TTT TCTGTC 938 Asp Ala Glu Phe Gly Asp Glu Gly Tyr Tyr Ser Cys Val Phe Ser Val155 160 165 CAC CAT AAT GGG ACA CGG TAC AAC ATC ACC AAG ACT GTC AAT ATAACA 986 His His Asn Gly Thr Arg Tyr Asn Ile Thr Lys Thr Val Asn Ile Thr170 175 180 GTT ATT GAA GGA AGG AGT AAA GTA ACT CCA GCT ATT TTA GGA CCAAAG 1034 Val Ile Glu Gly Arg Ser Lys Val Thr Pro Ala Ile Leu Gly Pro Lys185 190 195 200 TGT GAG AAG GTT GGT GTA GAA CTA GGA AAG GAT GTG GAG TTGAAC TGC 1082 Cys Glu Lys Val Gly Val Glu Leu Gly Lys Asp Val Glu Leu AsnCys 205 210 215 AGT GCT TCA TTG AAT AAA GAC GAT CTG TTT TAT TGG AGC ATCAGG AAA 1130 Ser Ala Ser Leu Asn Lys Asp Asp Leu Phe Tyr Trp Ser Ile ArgLys 220 225 230 GAG GAC AGC TCA GAC CCT AAT GTG CAA GAA GAC AGG AAG GAGACG ACA 1178 Glu Asp Ser Ser Asp Pro Asn Val Gln Glu Asp Arg Lys Glu ThrThr 235 240 245 ACA TGG ATT TCT GAA GGC AAA CTG CAT GCT TCA AAA ATA CTGAGA TTT 1226 Thr Trp Ile Ser Glu Gly Lys Leu His Ala Ser Lys Ile Leu ArgPhe 250 255 260 CAG AAA ATT ACT GAA AAC TAT CTC AAT GTT TTA TAT AAT TGCACC GTG 1274 Gln Lys Ile Thr Glu Asn Tyr Leu Asn Val Leu Tyr Asn Cys ThrVal 265 270 275 280 GCC AAC GAA GAA GCC ATA GAC ACC AAG AGC TTC GTC TTGGTG AGA AAA 1322 Ala Asn Glu Glu Ala Ile Asp Thr Lys Ser Phe Val Leu ValArg Lys 285 290 295 GAA ATA CCT GAT ATC CCA GGC CAT GTC TTT ACA GGA GGAGTA ACT GTG 1370 Glu Ile Pro Asp Ile Pro Gly His Val Phe Thr Gly Gly ValThr Val 300 305 310 CTT GTT CTC GCC TCT GTG GCA GCA GTG TGT ATA GTG ATTTTG TGT GTC 1418 Leu Val Leu Ala Ser Val Ala Ala Val Cys Ile Val Ile LeuCys Val 315 320 325 ATT TAT AAA GTT GAC TTG GTT CTG TTC TAT AGG CGC ATAGCG GAA AGA 1466 Ile Tyr Lys Val Asp Leu Val Leu Phe Tyr Arg Arg Ile AlaGlu Arg 330 335 340 GAC GAG ACA CTA ACA GAT GGT AAA ACA TAT GAT GCC TTTGTG TCT TAC 1514 Asp Glu Thr Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe ValSer Tyr 345 350 355 360 CTG AAA GAG TGT CAT CCT GAG AAT AAA GAA GAG TATACT TTT GCT GTG 1562 Leu Lys Glu Cys His Pro Glu Asn Lys Glu Glu Tyr ThrPhe Ala Val 365 370 375 GAG ACG TTA CCC AGG GTC CTG GAG AAA CAG TTT GGGTAT AAG TTA TGC 1610 Glu Thr Leu Pro Arg Val Leu Glu Lys Gln Phe Gly TyrLys Leu Cys 380 385 390 ATA TTT GAA AGA GAT GTG GTG CCT GGC GGA GCT GTTGTC GAG GAG ATC 1658 Ile Phe Glu Arg Asp Val Val Pro Gly Gly Ala Val ValGlu Glu Ile 395 400 405 CAT TCA CTG ATA GAG AAA AGC CGG AGG CTA ATC ATCGTT CTC AGC CAG 1706 His Ser Leu Ile Glu Lys Ser Arg Arg Leu Ile Ile ValLeu Ser Gln 410 415 420 AGT TAC CTG ACT AAC GGA GCC AGG CGT GAG CTC GAGAGT GGA CTC CAC 1754 Ser Tyr Leu Thr Asn Gly Ala Arg Arg Glu Leu Glu SerGly Leu His 425 430 435 440 GAA GCA CTG GTA GAG AGG AAG ATT AAG ATC ATCTTA ATT GAG TTT ACT 1802 Glu Ala Leu Val Glu Arg Lys Ile Lys Ile Ile LeuIle Glu Phe Thr 445 450 455 CCA GCC AGC AAC ATC ACC TTT CTC CCC CCG TCGCTG AAA CTC CTG AAG 1850 Pro Ala Ser Asn Ile Thr Phe Leu Pro Pro Ser LeuLys Leu Leu Lys 460 465 470 TCC TAC AGA GTT CTA AAA TGG AGG GCT GAC AGTCCC TCC ATG AAC TCA 1898 Ser Tyr Arg Val Leu Lys Trp Arg Ala Asp Ser ProSer Met Asn Ser 475 480 485 AGG TTC TGG AAG AAT CTT GTT TAC CTG ATG CCCGCA AAA GCC GTC AAG 1946 Arg Phe Trp Lys Asn Leu Val Tyr Leu Met Pro AlaLys Ala Val Lys 490 495 500 CCA TGG AGA GAG GAG TCG GAG GCG CGG TCT GTTCTC TCA GCA CCT TGA 1994 Pro Trp Arg Glu Glu Ser Glu Ala Arg Ser Val LeuSer Ala Pro * 505 510 515 520 GCTCCAGACG AGCTTGATGT CAAAAGCAAGTGAAGCGCTG CTAGAGGTCA TGCGTGTGCC 2054 TATTCACAGC GGTAGCTGTG GTTCAAAAGGCTGAATTTTG TGACTATACC CCCCACTCCC 2114 AGTTAGGAGA GTTGTCATCG GGTCATCACAGATGAAACAG AGCCTTGGTT GTGATCCTGA 2174 ACTCGCAGAG GGGGCCTTGG GATTCACAAGAAATCAGTTT GTTATTCTTT CTTCCTCTGG 2234 AGCAGTGATT CCCAACCTGT GGGTTGTGGCCCCTTTGGCA AACCTTTATC TCCAAAATAG 2294 ATGTACGCTA TGATTCATAA CTGTAGCCAACTCACAGTTA CAAAGTAGCA ACGAAAAAAG 2354 TTTTATGGTT GGGGGTTTCA CCACAGTGTGAAGAACTGTA TTAAAGGGTT GAAGCATTAG 2414 GAAGGTTGAG AACCGCTGGC CTAGAGCTGTCTGCCCAAAG CTTCTTGTGA CCTTGCAAGT 2474 GCCTGAGTGA AGCAAGAATA TTCTAGGGAAGTCTAGAGCA GAGACTGTGC TGAACAAACA 2534 CAGTAGATTT TAGGAAAACC AAACCAAACCAAATGAAAGG AAAGGAAACA GAAAAAAAAA 2594 CAAGAAGAAT GGGGATTCTT AAGTAATTTTTGTAACTCAT GACTTCATGT GCTATTTGAC 2654 TGACTTGAGA AAAGAAGGTA AATTCATTCAACATCTGCTG TCACAACAGC TGTGTGTGAA 2714 AACCTAGCAT CAGAAGAGAG TTGGGAGAGTTTGAGACTTC GCTTTGTTCT TCTATCAGCC 2774 AAGCTTCGAC ACATGAAGTT TATTTTATATGAAATATATT TTGTATTAAA TCTGCC 2830 537 amino acids amino acid linearprotein 4 Met His His Glu Glu Leu Ile Leu Thr Leu Cys Ile Leu Ile ValLys -18 -15 -10 -5 Ser Ala Ser Lys Ser Cys Ile His Arg Ser Gln Ile HisVal Val Glu 1 5 10 Gly Glu Pro Phe Tyr Leu Lys Pro Cys Gly Ile Ser AlaPro Val His 15 20 25 30 Arg Asn Glu Thr Ala Thr Met Arg Trp Phe Lys GlySer Ala Ser His 35 40 45 Glu Tyr Arg Glu Leu Asn Asn Arg Ser Ser Pro ArgVal Thr Phe His 50 55 60 Asp His Thr Leu Glu Phe Trp Pro Val Glu Met GluAsp Glu Gly Thr 65 70 75 Tyr Ile Ser Gln Val Gly Asn Asp Arg Arg Asn TrpThr Leu Asn Val 80 85 90 Thr Lys Arg Asn Lys His Ser Cys Phe Ser Asp LysLeu Val Thr Ser 95 100 105 110 Arg Asp Val Glu Val Asn Lys Ser Leu HisIle Thr Cys Lys Asn Pro 115 120 125 Asn Tyr Glu Glu Leu Ile Gln Asp ThrTrp Leu Tyr Lys Asn Cys Lys 130 135 140 Glu Ile Ser Lys Thr Pro Arg IleLeu Lys Asp Ala Glu Phe Gly Asp 145 150 155 Glu Gly Tyr Tyr Ser Cys ValPhe Ser Val His His Asn Gly Thr Arg 160 165 170 Tyr Asn Ile Thr Lys ThrVal Asn Ile Thr Val Ile Glu Gly Arg Ser 175 180 185 190 Lys Val Thr ProAla Ile Leu Gly Pro Lys Cys Glu Lys Val Gly Val 195 200 205 Glu Leu GlyLys Asp Val Glu Leu Asn Cys Ser Ala Ser Leu Asn Lys 210 215 220 Asp AspLeu Phe Tyr Trp Ser Ile Arg Lys Glu Asp Ser Ser Asp Pro 225 230 235 AsnVal Gln Glu Asp Arg Lys Glu Thr Thr Thr Trp Ile Ser Glu Gly 240 245 250Lys Leu His Ala Ser Lys Ile Leu Arg Phe Gln Lys Ile Thr Glu Asn 255 260265 270 Tyr Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Asn Glu Glu Ala Ile275 280 285 Asp Thr Lys Ser Phe Val Leu Val Arg Lys Glu Ile Pro Asp IlePro 290 295 300 Gly His Val Phe Thr Gly Gly Val Thr Val Leu Val Leu AlaSer Val 305 310 315 Ala Ala Val Cys Ile Val Ile Leu Cys Val Ile Tyr LysVal Asp Leu 320 325 330 Val Leu Phe Tyr Arg Arg Ile Ala Glu Arg Asp GluThr Leu Thr Asp 335 340 345 350 Gly Lys Thr Tyr Asp Ala Phe Val Ser TyrLeu Lys Glu Cys His Pro 355 360 365 Glu Asn Lys Glu Glu Tyr Thr Phe AlaVal Glu Thr Leu Pro Arg Val 370 375 380 Leu Glu Lys Gln Phe Gly Tyr LysLeu Cys Ile Phe Glu Arg Asp Val 385 390 395 Val Pro Gly Gly Ala Val ValGlu Glu Ile His Ser Leu Ile Glu Lys 400 405 410 Ser Arg Arg Leu Ile IleVal Leu Ser Gln Ser Tyr Leu Thr Asn Gly 415 420 425 430 Ala Arg Arg GluLeu Glu Ser Gly Leu His Glu Ala Leu Val Glu Arg 435 440 445 Lys Ile LysIle Ile Leu Ile Glu Phe Thr Pro Ala Ser Asn Ile Thr 450 455 460 Phe LeuPro Pro Ser Leu Lys Leu Leu Lys Ser Tyr Arg Val Leu Lys 465 470 475 TrpArg Ala Asp Ser Pro Ser Met Asn Ser Arg Phe Trp Lys Asn Leu 480 485 490Val Tyr Leu Met Pro Ala Lys Ala Val Lys Pro Trp Arg Glu Glu Ser 495 500505 510 Glu Ala Arg Ser Val Leu Ser Ala Pro 515 519 8 amino acids aminoacid single linear peptide NO NO FLAG peptide 5 Asp Tyr Lys Asp Asp AspAsp Lys 1 5

1-32. (Cancelled).
 33. An antibody that binds a polypeptide selectedfrom the group consisting of: a) a polypeptide consisting of aminoacids-19 to 522 of SEQ ID NO:2; b) a polypeptide consisting of aminoacids 1 to 522 of SEQ ID NO:2; c) a polypeptide consisting of aminoacids −19 to 310 of SEQ ID NO:2; d) polypeptide consisting of aminoacids 1 to 310 of SEQ ID NO:2; e) a polypeptide consisting of aminoacids 1 to 310 or SEQ ID NO:2, with the proviso that the polypeptidelacks the residue at position 298 of SEQ ID NO:2; f) a polypeptideconsisting of amino acids 1 to 522 of SEQ ID NO:2 with the proviso thatthe polypeptide lacks the residue at position 298 of SEQ ID NO:2; g)polypeptide consisting of amino acids −19 to 310 of SEQ ID NO:2, withthe proviso that the polypeptide lacks the residue at position 298 ofSEQ ID NO:2; and h) a polypeptide consisting of amino acids −19 to 522of SEQ ID NO:2, with the proviso that the polypeptide lacks the residueat position 298 of SEQ ID NO:2.
 34. An antibody that binds a polypeptideconsisting of amino acids 1 to 310 of SEQ ID NO:2.
 35. An antibody thatbinds a polypeptide consisting of amino acids 1 to 310 of SEQ ID NO:2,with the proviso that the polypeptide lacks the residue at position 298of SEQ ID NO:2.
 36. An antibody that binds a polypeptide consisting ofamino acids 1 to 522 of SEQ ID NO:2.
 37. An antibody that binds apolypeptide consisting of amino acids 1 to 522 of SEQ ID NO:2, with theproviso that the polypeptide lacks the residue at position 298 of SEQ IDNO:2.
 38. An antibody that binds a polypeptide consisting of amino acids−19 to 310 of SEQ ID NO:2.
 39. An antibody that binds a polypeptideconsisting of amino acids −19 to 310 of SEQ ID NO:2, with the provisothat the polypeptide lacks the residue at position 298 of SEQ ID NO:2.40. An antibody that binds a polypeptide consisting of amino acids −19to 522 of SEQ ID NO:2.
 41. An antibody that binds a polypeptideconsisting of amino acids −19 to 522 of SEQ ID NO:2, with the provisothat the polypeptide lacks the residue at position 298 of SEQ ID NO:2.42. A method for treating rheumatoid arthritis in an individual, themethod comprising administering to the individual a compositioncomprising an antibody that binds a polypeptide consisting of aminoacids 1-310 of SEQ ID NO:2.
 43. A method for treating rheumatoidarthritis in an individual, the method comprising administering to theindividual a composition comprising an antibody that binds a polypeptideconsisting of amino acids 1-310 if SEQ ID NO:2, with the proviso thatthe polypeptide lacks the residue at position 298 of SEQ ID NO:2.
 44. Amethod for treating inflammatory bowel disease in an individual, themethod comprising administering to the individual a compositioncomprising an antibody that binds a polypeptide consisting of aminoacids 1-310 of SEQ ID NO:2.
 45. A method for treating inflammatory boweldisease in an individual, the method comprising administering to theindividual a composition comprising an antibody that binds a polypeptideconsisting of amino acids 1-310 of SEQ ID NO:2, with the proviso thatthe polypeptide lacks the residue at position 298 of SEQ ID NO:2.